The most important work we can do, individually and globally, is the healing of traumas so that we don’t pass them down to future generations. This blog is a working tool to contribute to this good work.

Department of Behavioral Sciences, College of Judea and Samaria, Ariel, Israel. fride@research.yosh.ac.il

The appetite-stimulating effects of the cannabis plant (Cannabis sativa)

…..have been known since ancient times,

….and appear to be effected through the incentive and rewarding properties of foods.

Investigations into the biological basis of the multiple effects of cannabis have yielded important breakthroughs in recent years:

…..the discovery of two cannabinoid receptors in brain and peripheral organ systems,

…..and endogenous ligands (endocannabinoids) for these receptors.

……These advances have greatly increased our understanding of how appetite is regulated through these endocannabinoid receptor systems.

The presence of endocannabinoids in the developing brain and in maternal milk have led to evidence for a critical role for CB1 receptors in oral motor control of suckling during neonatal development.

The endocannabinoids appear to

….regulate energy balance

….and food intake

….at four functional levels within the brain and periphery:

(i) limbic system (for hedonic evaluation of foods),

(ii) hypothalamus and hindbrain (integrative functions),

(iii) intestinal system, and

(iv) adipose tissue.

At each of these levels,

….the endocannabinoid system interacts

….with a number of better known molecules involved in appetite and weight regulation,

…including leptin, ghrelin, and the melanocortins.

Therapeutically, appetite stimulation by cannabinoids has been studied for several decades, particularly in relation to cachexia and malnutrition associated with cancer, acquired immunodeficiency syndrome, or anorexia nervosa. The recent advances in cannabinoid pharmacology may lead to improved treatments for these conditions or,

conversely, for combating excessive appetite and body weight, such as CB1 receptor antagonists as antiobesity medications.

In conclusion, the exciting progress in the understanding of how the endocannabinoid CB receptor systems influence appetite and body weight is stimulating the development of therapeutic orexigenic and anorectic agents.

Furthermore, the role of cannabinoid CB1 receptor activation for milk suckling in newborns may open new doors toward understanding nonorganic failure-to-thrive in infants, who display growth failure without known organic cause.

Endogenous [our body’s own] cannabinoids (endocannabinoids) and their cannabinoid CB1 and CB2 receptors, are present from the early stages of gestation and play a number of vital roles for the developing organism.

Although most of these data are collected from animal studies, a role for cannabinoid receptors in the developing human brain has been suggested…

In addition, a role for the endocannabinoid system for the human infant is likely, since they have been detected in human milk.

Animal research indicates that the Endocannabinoid system fulfills a number of roles in the developing organism:

1. embryonal implantation

2. in neural development

3. as a neuroprotectant (…protects the developing brain from trauma-induced neuron [cell] loss)

4. in the initiation of suckling in the newborn (working in the neonatal brain and critical for survival).

5. In addition, subtle but definite deficiencies have been described in memory, motor and addictive behaviors and in higher cognitive (‘executive’) function in the human offspring as result of prenatal exposure to marihuana.

Therefore, the endocanabinoid-CB1 receptor system may play a role in the development of structures which control these functions

From the multitude of roles of the endocannabinoids and their receptors in the developing organism, there are two distinct stages of development, during which proper functioning of the endocannabinoid system seems to be critical for survival:

…….embryonal implantation and

………neonatal milk sucking.

Dysfunctions in the Endocannabinoid system in infants with growth failure resulting from an inability to ingest food, may resolve the enigma of “non-organic failure-to-thrive” (NOFTT). Developmental observations suggest further that CB1 receptors develop only gradually during the postnatal period, which correlates with an insensitivity to the psychoactive effects of cannabinoid treatment in the young organism.

Therefore, it is suggested that children may respond positively to medicinal applications of cannabinoids without undesirable central effects.

………Excellent clinical results have previously been reported in pediatric oncology and in case studies of children with severe neurological disease or brain trauma.

………..We suggest cannabinoid treatment for children or young adults with cystic fibrosis in order to achieve an improvement of their health condition including improved food intake and reduced inflammatory exacerbations.

Department of Behavioral Sciences, College of Judea and Samaria, Ariel 44837, Israel. fride@research.yosh.ac.il

Endogenous cannabinoids (endocannabinoids) and their cannabinoid CB1 and CB2 receptors,

……are present from the early stages of gestation

……and play a number of vital roles for the developing organism.

Although most of these data are collected from animal studies, a role for cannabinoid receptors in the developing human brain has been suggested, based on the detection of “atypically” distributed CB1 receptors in several neural pathways of the fetal brain.

In addition, a role for the endocannabinoid system for the human infant is likely, since the endocannabinoid 2-arachidonoyl glycerol has been detected in human milk.

Animal research indicates that the Endocannabinoid-CB1 Receptor (‘ECBR’) system fulfills a number of roles in the developing organism:

2. in neural development (by the transient presence of CB1 receptors in white matter areas of the nervous system);

3. as a neuroprotectant (anandamide protects the developing brain from trauma-induced neuronal loss);

4. in the initiation of suckling in the newborn (where activation of the CB1 receptors in the neonatal brain is critical for survival).

5. In addition, subtle but definite deficiencies have been described in memory, motor and addictive behaviors and in higher cognitive (‘executive’) function in the human offspring as result of prenatal exposure to marihuana.

Therefore, the endocanabinoid-CB1 receptor system may play a role

in the development of structures which control these functions, including the nigrostriatal pathway and the prefrontal cortex.

From the multitude of roles of the endocannabinoids and their receptors in the developing organism, there are two distinct stages of development, during which proper functioning of the endocannabinoid system seems to be critical for survival:

…….embryonal implantation and

………neonatal milk sucking.

We propose that a dysfunctional Endocannabinoid-CB1 Receptor system in infants with growth failure resulting from an inability to ingest food, may resolve the enigma of “non-organic failure-to-thrive” (NOFTT). Developmental observations suggest further that CB1 receptors develop only gradually during the postnatal period, which correlates with an insensitivity to the psychoactive effects of cannabinoid treatment in the young organism.

Therefore, it is suggested that children may respond positively to medicinal applications of cannabinoids without undesirable central effects.

………Excellent clinical results have previously been reported in pediatric oncology and in case studies of children with severe neurological disease or brain trauma.

………..We suggest cannabinoid treatment for children or young adults with cystic fibrosis in order to achieve an improvement of their health condition including improved food intake and reduced inflammatory exacerbations.

Fride 2004b

Abstract – Israel

Recent research suggests that the endogenous cannabinoids (“endocannabinoids”) and their cannabinoid receptors have a major influence during pre- and postnatal development.

First, high levels of the endocannaboid anandamide and cannabinoid receptors

….are present in the preimplantation embryo and in the uterus,

…..while a temporary reduction of anandamide levels is essential for embryonal implantation.

In women accordingly, an inverse association has been reported between fatty acid amide hydrolase (the anandamide degrading enzyme) in human lymphocytes and miscarriage.

Second, CB(1) receptors display a transient presence

….in white matter areas of the pre- and postnatal nervous system, …..suggesting a role for CB(1) receptors in brain development.

Third, endocannabinoids have been detected in maternal milk

….and activation of CB(1) receptors appears to be critical for milk sucking by newborn mice,

…….apparently activating oral-motor musculature.

Fourth, anandamide has neuroprotectant properties in the developing postnatal brain.

The consumption by women of cannabis derivatives during pregnancy and/or lactation

….affects the development of their offspring

….because like other psychoactive drugs, cannabinoids,

…..the psychoactive ingredients of marijuana,

…..can cross the placental barrier and be secreted into the maternal milk.

Through this way, cannabinoids

….are able to affect the expression of key genes for neural developmental ……leading to neurotransmitter and behavioral disturbances.

In this present study, we wanted to explore the influence of prenatal cannabinoid exposure

….on the gene expression of a key protein for brain development,

…..the neural adhesion molecule L1,

…..which plays an important role in processes of cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis.

Our results confirmed that the levels of L1 transcripts were significantly increased after prenatal delta9-THC exposure in several regions such as

..the fimbria,

…stria terminalis,

…stria medullaris and

…corpus callosum,

….which share the properties of being white matter regions and containing, exclusively during development,

……….an abundant population of cannabinoid CB1 receptors, the major targets for the action of plant-derived cannabinoids.

So THC can cross the placenta barrier and also find their way through the milk not only to the mother’s receptors but also to those in the infant

L1-mRNA levels were also increased in grey matter structures such as the ….septum nuclei and

….the habenula,

….but remained unchanged in most of the grey matter structures analyzed (cerebral cortex, basolateral amygdaloid nucleus, hippocampus, thalamic and hypothalamic nuclei, basal ganglia and subventricular zones) and also in a few white matter structures (fornix and fasciculus retroflexus).

An important aspect of these observations is that the increase in L1-mRNA levels reached statistical significance

….only in the case of delta9-THC-exposed males

…..but not in the case of delta9-THC-exposed females where only trends or no effects were detected,

…..this supporting previous evidence on a sexual dimorphism, with greater effects in male fetuses, for the action of cannabinoids in the developing brain.

In summary, cannabinoids seem to influence the expression of L1 in specific brain structures during the prenatal period, which, considering the

………….role played by this protein in different events related to neural development, might explain the neurotransmitter and behavioral disturbances reported after prenatal consumption of marijuana.

At the same concentrations, the membrane-impermeant conjugate of progesterone with BSA was ineffective, suggesting that binding to an intracellular receptor was needed for progesterone activity.

Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level, and was partly mediated by the Th2 cytokines.

In fact, lymphocyte treatment with IL-4 or with IL-10 had a stimulating effect on FAAH, whereas the

Th1 cytokines IL-12 and IFN-gamma reduced the activity and the protein expression of FAAH. Human chorionic gonadotropin or cortisol had no effect on FAAH activity. At variance with FAAH, the lymphocyte anandamide transporter and cannabinoid receptors were not affected by treatment with progesterone or cytokines. Good FAAH substrates such as anandamide and 2-arachidonoylglycerol inhibited the release of leukemia-inhibitory factor from human lymphocytes, but N-palmitoylethanolamine, a poor substrate, did not. A clinical study performed on 100 healthy women showed that

a low FAAH activity in lymphocytes correlates with spontaneous abortion,

whereas anandamide transporter and cannabinoid receptors in these cells remain unchanged. These results

add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.

………..This would support the notion that the cannabinoid system might play a modulatory role in the regulation of these processes.

We have recently presented preliminary in vivo evidence showing that this modulatory action might be exerted, among others, through regulating the levels of several key elements in these processes, such as the L1 protein. This was observed in various white matter areas of the rat forebrain. Because these preliminary in vivo experiments focused only in fetal ages, we concentrated now in the period of early postnatal development.

To this end, we analyzed the effects of the cannabinoid agonist Delta(9)-tetrahydrocannabinol (Delta(9)-THC) daily administered since the 5th day of gestation on mRNA levels for L1 in different brain structures of rat neonates at different postnatal ages (PND1, PND5 and PND12). Our results revealed that Delta(9)-THC exposure affected the levels of L1 transcripts in specific brain structures only in PND1, these effects disappearing during further days. Thus, we found reduced L1-mRNA levels in grey matter regions, such as the cerebral cortex, septum nuclei, striatum, dentate gyrus and CA3 subfield of the Ammon horn. White matter areas and subventricular zones were, however, more resistant to Delta(9)-THC exposure at this postnatal age in contrast with the previous data obtained in the fetal brain. Importantly, the effects were influenced by gender of animals, since the reductions were always more marked in females than males, also in contrast with the data reported for the fetal brain.

In summary, the cannabinoid system seems to modulate the levels of L1 in several brain structures during specific periods of development [late gestation (previous data) and very early postnatal days (present data)], which correlates with the periods in which we had previously found an atypical distribution of CB(1) receptors in the developing brain. However, the magnitude of the effects of cannabinoids on L1 was influenced by two factors: gender and age of development.

Considering the role played by L1 in different events related to neural development,

….our observations might support the occurrence of a physiological mechanism

……by which the cannabinoid system might regulate processes

…….such as cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis.

The use of marijuana, which today is the most used recreational drug, has been demonstrated to affect adversely reproduction. Marijuana smokers, both men and women, show impaired fertility, owing to defective signaling pathways, aberrant hormonal regulation, or wrong timing during embryo implantation. ….These ‘endocannabinoids’ exert several actions either in the central nervous system or in peripheral tissues, and are metabolized by specific enzymes that synthesise or hydrolyze them. In this review, we shall describe the elements that constitute the endocannabinoid system (ECS), in order to put in a better perspective the role of this system in the control of human fertility, both in females and males. In addition, we shall discuss the interplay between ECS, sex hormones and cytokines, which generates an endocannabinoid-hormone-cytokine array critically involved in the control of human reproduction.

Ambulatory activity between WIN 55,212-2- and vehicle-treated adult offspring did not vary.

……..Our results show that prenatal exposure to cannabinoid agonist induces a long-term alteration of endocannabinoid system in brain areas involved in learning-memory, motor activity and emotional behavior

This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adolescents and adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

We also show that the non-hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. ………. We show that

sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization.

These observations open new perspectives on the understanding and treatment of male fertility problems.

Division of Anatomy and Cell Biology, Department of Pathology and Anatomical Sciences, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA. schuel@acsu.buffalo.edu

Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs.

Arachidonylethanolamide (AEA), also known as anandamide, is a novel lipid-signal molecule that is an endogenous agonist (endocannabinoid) for cannabinoid receptors.

….Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct.

to human sperm … express cannabinoid receptors.

R-methanandamide [AM-356], a potent and metabolically stable AEA analog, and (-)delta(9) tetrahydrocannabinol (THC), the major psychoactive constituent of Cannabis, modulated capacitation and fertilizing potential of human sperm in vitro.

AM-356 elicited biphasic effects on the incidence of hyperactivated sperm motility (HA) between 1 and 6 hr of incubation: at (2.5 nM) it inhibited HA, while at (0.25 nM) it stimulated HA. …Sperm fertilizing capacity… was reduced 50% by (1 nM) AM-356.

These findings suggest that AEA-signaling may regulate sperm functions required for fertilization in human reproductive tracts,

…..and imply that smoking of marijuana could impact these processes.

This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

Division of Anatomy & Cell Biology, Department of Pathology & Anatomical Sciences, School of Medicine & Biomedical Sciences, University at Buffalo, State University of New York, 14214, USA. schuel@acsu.buffalo.edu

The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids.

These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm.

Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy.

The endocannabinoid system and the presence of CB1 receptor (CB1-R) target of the anandamide were identified in human sperm, however the anandamide action in this context needs to be further elucidated. At this purpose we analyzed the effects of anandamide on human sperm capacitation and motility. Afterwards, we focused on lipid and glucose sperm metabolism and also investigated the interrelationship between anandamide and insulin secretion by sperm. By intracellular free Ca(2+) content assay and proteins tyrosine phosphorylation, we evidenced that anandamide did not induce capacitation process and a negative effect was obtained on sperm motility. The blockage of CB1-R by the specific antagonist SR141716 increased both capacitation and sperm motility suggesting an involvement of the CB1-R in the acquisition of sperm fertilizing activity. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities, suggest that anandamide exerts a lipogenetic effect on human sperm lipid metabolism. Concerning the glucose metabolism, anandamide increases GSK3 phosphorylation indicating that it is involved in the accumulation of energy substrates. G6PDH activity was not affected by anandamide. Interestingly, AEA is involved in insulin secretion by sperm. As insulin had been demonstrated to be an autocrine factor that triggers capacitation, the endocannabinoid might be inserted in the signaling cascade that induces this process. Altogether these findings highlight a pivotal involvement of the CB1-R in the control of sperm energy homeostasis and propose a new site of action for endocannabinoids in the control of energy metabolism. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc.

Since the end of the 1970s, studies have shown that, besides the endocrine route, a chemical mediator may also act through autocrine and/or paracrine mechanisms. This has opened new frontiers for research as a result of a redefinition of what endocrinology represents. Apart from androgens within the male gonad, testicular gonadotropin-releasing hormone, estrogens, molecular chaperones, proto-oncogenes, and, very recently, the endocannabinoid system have been shown to play important roles. Their activities to regulate spermatogenesis, including spermiogenesis and sperm maturation, will be discussed from the comparative viewpoint to describe adaptive phenomena and to speculate on evolution.

The recent identification and cloning of guanine nucleotide regulatory protein-coupled brain-type and spleen-type cannabinoid receptors (CB1-R and CB2-R, respectively) provide evidence that many of the effects of cannabinoids are mediated via these receptors.

……….Our recent observation of expression of both CB1-R and CB2-R genes in the preimplantation mouse embryo suggests that it could also be a target for cannabinoids.

……..Indeed, cannabinoid agonists interfered with preimplantation embryo development in vitro.

… it is apparent that the mouse blastocyst has many more high-affinity receptors than those in the mouse brain

…. Cannabinoid agonists and the CB1-R antagonist SR141716A effectively competed for anandamide binding in the blastocyst.

…. To determine whether cannabinoid inhibition of embryonic development could be reversed by SR141716A, 2-cell embryos were cultured in the presence of cannabinoid agonists with or without SR141716A for 72 h. Most of the 2-cell embryos cultured in the absence of the agonists developed into blastocysts (approximately 90%). I

………n contrast, the addition of cannabinoid agonists anandamide, Win 55212-2, or CP 55,940 in the culture medium severely compromised embryonic development: more than 60% of the 2-cell embryos failed to develop to blastocysts.

……… A reduction in trophectoderm cell numbers was noted in those blastocysts that escaped the developmental arrest in the presence of cannabinoid agonists. However, this reduction was corrected when embryos were cultured simultaneously with an agonist and SR141716A. Furthermore, embryonic arrest was reversed when embryos were cultured simultaneously in the presence of an agonist and SR141716A. The addition of SR141716A alone in the culture medium apparently had no effects on embryonic development: more than 90% of the embryos developed into blastocysts. The results suggest that the CB1 receptors in preimplantation mouse embryos are biologically active and cannabinoid effects on them are primarily mediated by these receptors.

Arachidonoylethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors. We demonstrated previously that ligand-receptor signaling with cannabinoids is operative in both the mouse embryo and uterus during the periimplantation period. In the present investigation, we provide evidence that

….. mouse uterus has the enzymatic capacities to form (synthase) and hydrolyze (amidase) anandamide.

……..These activities were primarily localized in uterine microsomes and were dependent upon pH, time, protein, and substrate concentrations.

While the synthase showed maximal activity at pH 9.0, the amidase activity was maximal at pH 8.5.

To determine whether uterine synthase and/or amidase activity have any physiological significance with respect to uterine receptivity and implantation during early pregnancy, profiles of synthase and amidase activities were analyzed in mouse uterine microsomes obtained during early pregnancy or pseudopregnancy. It should be noted that the

synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to the receptive state are critical to the embryo implantation process.

In the mouse, the uterus becomes receptive for implantation only for a limited period during pregnancy or pseudopregnancy. The uterus becomes receptive on day 4 (the day of implantation) and by day 5, it becomes nonreceptive for blastocyst implantation (Paria et al., 1993: Proc Natl Acad Sci USA 90:10159-10162.).

Both anandamide synthase and amidase activities remained virtually unaltered on days 1-4 of pregnancy. In contrast, while the synthase activity increased, the amidase activity decreased in the uterus on day 5 of pseudopregnancy (nonreceptive phase) as compared to those observed on day 4 of pregnancy or pseudopregnancy (receptive phase). The synthase and amidase activities in surgically separated implantation and interimplantation sites showed an interesting profile on days 5-7 of pregnancy; the synthase activity was lower in implantation sites as compared to that in interimplantation sites.

In contrast, amidase activity was higher in implantation sites compared with that in interimplantation sites.

Since we have shown previously that cannabinoids including anandamide interfere with preimplantation mouse embryo development,

….the local modulation of anandamide formation and hydrolysis

…by the implanting blastocysts could be critical for successful embryonic growth, implantation, and pregnancy establishment. Which way does this go? I think later research shows that the change occurs on the uterine site, and is not controlled by the blastocyst

The finding of increased synthase activity with concomitant

….decrease in amidase activity in the uterus on day 5 of pseudopregnancy, when

……..the uterus is hostile to blastocyst survival and implantation,

is consistent with this assumption. Further indomethacin, known to interfere with arachidonate metabolism and embryo implantation, stimulated the synthase activity, while inhibiting the amidase activity in the uterus in vivo and in vitro. Finally, considering the kinetics and profiles of these two enzymatic reactions during early pregnancy, the results suggest that synthase and amidase may be two separate enzymes in the mouse uterus. This investigation constitutes the first detailed studies on anandamide synthase and amidase activities in the female reproductive tract

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors.

Department of Molecular and Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City 66160-7338, USA.

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that

Mammalian conception is a complex process regulated by both sexual behavior and reproductive performance. Alcohol, marijuana and tobacco are among the main factors which affect negatively fertility in women and men. Several studies have demonstrated that marijuana impairs the male copulatory activity, and that smokers of this illegal drug show reduced fertility due, for instance, to decrease in sperm concentration, defective sperm function or alteration of sperm morphology.The discovery of endocannabinoids and all components responsible for their metabolism has allowed [us] to collect valuable information on the effects of these endogenous lipids, able to mimic the actions of delta-9-tetrahydrocannabinol (THC), in reproductive functions.

The purpose of this review is to describe the actions of cannabinoids and endocannabinoids on the control of procreation and hormonal release during the fertilization process in males.

Cannabinoids, the main active components of marijuana, have been shown to exert different adverse effects on male reproduction both in vertebrates and invertebrates. The main effects of endocannabinoids, a particular group of endogenously produced cannabinoids, in sperm are the inhibition of motility, capacitation and acrosome reaction, all fundamental processes necessary for oocyte penetration, whose alteration leads to the inhibition of sperm fertilizing ability. These inhibitory effects are mediated by the direct action of endocannabinoids on sperm through the activation of the cannabinoid receptor subtype 1 that has been shown to be expressed in mature sperm. In many different cell types it has been demonstrated that endocannabinoids negatively influence mitochondrial activity. In the present paper it will be briefly reviewed the role of endocannabinoids, on sperm motility, capacitation and acrosome reaction with particular attention on the possible interference of endocannabinoids with sperm mitochondrial activity.

Cannabinoids have been always identified as harmful drugs because of their negative effects on male and female reproduction. The discovery of the ‘endocannabinoid system (ECS)’ … has enabled a wealth of information on the significance of endocannabinoid signaling in multiple reproductive events: Sertoli cell survival, spermatogenesis, placentation, fertilization, preimplantation embryo development, implantation and postimplantation embryonic growth. These studies have also opened new perspectives in clinical applications, pointing to the ECS as a new target for correcting infertility and for improving reproductive health in humans. This review will focus on the involvement of type-2 cannabinoid (CB2) receptors in reproductive biology, covering both the male and female sides. It will also discuss the potential relevance of the

immunological activity of CB2 [receptors] at the maternal/foetal interface,

An emerging concept in female reproduction is the role of endocannabinoids,

…..a group of endogenously produced lipid mediators that bind to and activate cannabinoid receptors.

Although adverse effects of cannabinoids in female reproduction have been implicated for years, the mechanisms by which they exert these effects remained elusive.

….With the identification of cannabinoid receptors, endocannabinoid ligands, their key synthetic and hydrolytic pathways, and the generation of knockout mouse models for cannabinoid receptors, a wealth of information is now available regarding the

…. significance of cannabinoid/endocannabinoid signaling in early pregnancy.

This review focuses on various aspects of endocannabinoid signaling in preimplantation embryo development and activation, and uterine differentiation during the periimplantation embryo-uterine dialog. It is hoped that a deeper understanding will lead to potential clinical applications of the endocannabinoid system as a target for regulating female fertility.

Linda note: Talk about an interrupted attachment system! Sometimes this research process just seems too strange to me – like it is mandated from “somewhere else” and I am just a part in it, and I don’t know why — just that I suspect that even this, just past how the sperm joins the egg when fertilization occurs – which is not really “an attachment” so much as a joining – the implantation of the zygote to the mother in the womb is where attachment begins. I suspected that the opioid system is at the root of the attachment system – but I don’t know how to think about this information on the cannabinoid system – How can it be so important and central and “we” the public know nothing about it? Sometimes this just gets very intense! And somehow deeply spiritual – and much bigger than I am, and significant somehow – though I don’t know how.

And how is this tied to trauma – except a failure to attach to the mother certainly creates a fundamental trauma problem for the zygote, who is going to pass in and out of this world with only the potential for a voice or a finger print.

There are reports of adverse effects of cannabinoids on pregnancy outcome including retarded embryo development and pregnancy failure. Thus, discoveries of endogenous cannabinoid-like lipid mediators and cannabinoid receptors raise questions about their pathophysiological roles during normal pregnancy.

……….We previously reported that anandamide,

……… an endogenously produced arachidonate derivative (endocannabinoid), is

………synthesized in the female reproductive tracts, and it

………acts on cannabinoid receptors expressed

……..on the cell surface of the embryo

……..to regulate the preimplantation embryo development and implantation in mice.

Anandamide (N -arachidonoylethanolamine, AEA) is a major endocannabinoid, shown to impair mouse pregnancy and embryo development and to induce apoptosis [cell death] in blastocysts. Here, we review the roles of AEA, of the AEA-binding cannabinoid (CB) receptors, of the selective AEA membrane transporter (AMT), and of the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), in human gestation. In particular, we discuss the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, showing that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy.

In this line, we discuss the critical role of FAAH in human peripheral lymphocytes, showing that the expression of this enzyme is regulated by progesterone, Th1 and Th2 cytokines, which also regulate fertility.Moreover, we show that

AEA and the other endocannabinoid, 2-arachidonoylglycerol,

inhibit the release of the fertility-promoting cytokine leukemia inhibitory factor from human lymphocytes.

Taken together, low FAAH and consistently high blood levels of AEA, but not CB receptors or AMT, can be early (<8 weeks of gestation) markers of spontaneous abortion, potentially useful as diagnostic tools for large-scale, routine monitoring of gestation in humans.

The endocannabinoid system, including its receptors (CB(1) and CB(2)), endogenous ligands (‘endocannabinoids’), synthesizing and degrading enzymes, as well as transporter molecules,

…….has been detected from the earliest stages of embryonic development ….and throughout pre- and postnatal development.

In addition, the endocannabinoids,

notably 2-arachidonyl glycerol,

are also present in maternal milk.

During three distinct developmental stages

….(i.e. embryonic implantation,

…..prenatal brain development and

…..postnatal suckling),

……the endocannabinoid system appears to play an essential role for development and survival

. Thus, during early pregnancy, successful embryonic passage through the oviduct and implantation into the uterus

…….both require critical enzymatic control of optimal anandamide levels at the appropriate times and sites.

During foetal life, the cannabinoid CB(1) receptor plays a major role in brain development, regulating neural progenitor differentiation into neurones and glia and guiding axonal migration and synaptogenesis.

Postnatally, …a critical role for CB(1) receptors in the initiation of milk suckling by neonates,

………possibly by interfering with innervation of the tongue muscles.

Manipulating the endocannabinoid system by pre- and/or postnatal administration of cannabinoids or maternal marijuana consumption, has significant, yet subtle effects on the offspring.

Thus, alterations in the dopamine, GABA and endocannabinoid systems have been reported while enhanced drug seeking behaviour and impaired executive (prefrontal cortical) function have also been observed.

……….The relatively mild nature of the disruptive effects of prenatal cannabinoids may be understood in the framework of the

………..intricate timing requirements and frequently biphasic effects of the (endo)cannabinoids.

In conclusion, the endocannabinoid system plays several key roles in pre- and postnatal development.

There is recent evidence supporting the notion that the cannabinoid signaling system

…plays a modulatory role in the regulation of cell proliferation and migration,

….survival of neural progenitors, neuritic elongation and guidance, and synaptogenesis.

………..This assumption is based on the fact that

……….cannabinoid 1-type receptors (CB(1) receptors) and

……..their ligands emerge early in brain development and

………are abundantly expressed in certain brain regions that play key roles in these processes.

We have recently presented in vivo evidence showing that this modulatory action might be exerted through regulating the synthesis of the cell adhesion molecule L1 that is also a key element for those processes. To further explore this issue, we conducted here immunohistochemical studies aimed at determining the cellular substrates of CB(1) receptor-L1 interactions in the rat brain during late fetal development. In this period, we previously found that the activation of CB(1) receptors increased L1 synthesis in several forebrain white matter regions but not in gray matter areas. Using double labeling studies, we observed here colocalization of both proteins in fiber tracts including the corpus callosum, the adjacent subcortical white matter, the internal capsule and the anterior commissure. Experiments conducted with cultures of fetal rat cortical nerve cells revealed that L1 is present mainly in neurons but not in glial cells. This fact, together with the results obtained in the double labeling studies, would indicate that

….. L1 and CB(1) receptors should possibly be present in axons elongating through these white matter tracts, or, alternatively, in migrating neurons.

……. Further experiments confirmed the presence of CB(1) receptors in elongating axons, since these receptors colocalized with growth-associated protein 43 (GAP-43), a marker of growth cones, but not with synaptophysin, a marker of active synaptic terminals, in the same forebrain white matter regions.

……….Lastly, using cultured fetal rat cortical neurons, we also observed that the activation of cannabinoid receptors increased the levels of the full-length L1 and altered those of some active proteolytic fragments of this protein whose generation has been associated with specific steps in the process of neuritic elongation in cultured neurons.

………. In summary, we have demonstrated that

………. the effects caused by cannabinoid agonists on L1 are facilitated by the colocalization of this cell adhesion molecule with CB(1) receptors in several forebrain white matter regions during fetal brain development.

………We have provided strong evidence that this phenomenon occurs in axons elongating through these white matter tracts,

……….and we have explored in vitro how cannabinoid receptors influence L1 levels.

………Considering the role played by L1 in different events related to neural development,

………our observations support the occurrence of a physiological mechanism

……..by which the cannabinoid system

……..might regulate the process of axonal growth and guidance ……..through regulating the synthesis and function of L1.

Brain lesions induced in newborn mice or rats by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) or S-bromowillardiine (acting on AMPA-kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage associated with cerebral palsy. Exogenous and endogenous cannabinoids have received increasing attention as potential neuroprotective agents in a number of neurodegenerative disorders of the adult.

One recent study showed neuroprotection by the cannabinoid agonist WIN-55212 in a newborn rat model of acute severe asphyxia. The present study was designed to assess the neuroprotective effects of the endogenous cannabinoid anandamide using a well-defined rodent model of neonatal excitotoxic brain lesions. In this model, anandamide provided dose-dependent and long-lasting protection of developing white matter and cortical plate reducing the size of lesions induced by S-bromowillardiine.

Real-time PCR confirmed the expression of CB1 receptors, but not CB2 receptors, in the untreated newborn neocortex.

Finally, neuroprotective effects of anandamide in white matter involved increased survival of preoligodendrocytes and better preservation of myelination. The present study provides experimental support for the role of endocannabinoids as a candidate therapy for excitotoxic perinatal brain lesions.

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal day 9-10] show behavioral alterations that resemble specific symptoms of schizophrenia. Accordingly, this experimental procedure has been proposed as an animal model of schizophrenia based on the neurodevelopmental hypothesis. We have recently reported that MD-induced sex-dependent alterations in the hippocampus of neonatal rats.

In view of recent evidence for

important implications of the cerebellum in neurodevelopmental psychiatric diseases, we have now addressed possible degenerative changes in the cerebellar cortex

of neonatal Wistar rats of both genders. To evaluate the presence of degenerated nerve cells, we used Fluoro-Jade C staining and for the study of astrocytes, we employed glial fibrillary acidic protein. Further, we analyzed the modulatory actions of two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N-arachidonoyl-serotonin, AA-5-HT, and the endocannabinoid reuptake inhibitor, OMDM-2 (daily subcutaneous injections during the postnatal period 7-12). The animals were sacrificed at postnatal Day 13.

MD induced significant increases in the number of

Fluoro-Jade C positive cells (indicative of degenerating neurons)

and in the number of glial fibrillary acidic protein positive cells, only in males.

The two cannabinoid compounds reversed or attenuated these effects. The present results provide new insights regarding the psychopathological implications of the cerebellum, the role of the endocannabinoid system in neural development, and the possible neurodevelopmental basis of gender differences in schizophrenia

My guess is that they don’t have to limit these effects only to schizophrenia!

Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation,

These results suggest that a tight regulation of endocannabinoid signaling

……..is important

……..for synchronizing embryo development

…..with uterine receptivity for implantation.

Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so in CB1(-/-)/CB2(-/-) double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.

……is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors.

To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that

…..14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R.

However, 56 nM anandamide is capable of

………inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that

………anandamide may play an essential role at the outset of implantation.

In the postnatal brain, endocannabinoids acting as retrograde messengers regulate the function of many synapses.

By contrast, the understanding of endocannabinoid functions

that regulate fundamental developmental processes

such as cell proliferation, migration, differentiation and survival

during patterning of the CNS is just beginning to unfold.

I bet this system is the gateway for stress changes in the postnatal developing brain! Those skeleton people need to read this information! Maybe this system does not fly solo here as they say it does in bone remodeling, but this is pretty important participation!

Increasing the knowledge of basic developmental and signaling principles that are controlled by endocannabinoids

will provide important insights into the molecular mechanisms

that establish functional neuronal circuits in the brain.

Moreover, determining the molecular basis of permanent modifications to cellular structure and intercellular communication imposedby cannabis smoking during pregnancywill provide novel therapeutic targets for alleviating pathogenic changes in affected offspring. Here, we summarize recent findings regarding the ontogeny of the endocannabinoid system in neurons that sculpt the temporal and spatial diversity of cellular functions during CNS development.

However, eCB functions during pyramidal cell specification and establishment of long-range axonal connections are unknown. Here, we show that eCB signaling is operational in subcortical proliferative zones from embryonic day 12 in the mouse telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells.

When layer patterning is accomplished, developing pyramidal cells rely on eCB signaling to initiate the elongation and fasciculation of their long-range axons.

Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, United Kingdom. t.harkany@abdn.ac.uk

Endocannabinoids (eCBs) regulate a broad range

of physiological functions in the postnatal brain

and are implicated in the

neuropathogenesis of psychiatric and metabolic diseases.

Accumulating evidence indicates that eCB signaling

also serves key functions during neurodevelopment; and is inherently involved in the

control of neurogenesis,

neural progenitor proliferation,

lineage segregation, and the

migration and phenotypic specification

of immature neurons.

Recent advances in developmental biology define fundamental eCB-driven cellular mechanisms that also contribute to our understanding of the molecular substrates of prenatal drug, in particular cannabis, actions. Here, we summarize known organizing principles of eCB-signaling systems in the developing telencephalon, and outline the sequence of decision points and underlying signaling pathways upon CB1 cannabinoid receptor activation that contribute to neuronal diversification in the developing brain. Finally, we discuss how these novel principles affect the formation of complex neuronal networks.